1. Field of the Invention
The present invention relates to novel inducible promoters derived from plants and their application in the controlled expression of heterologous genes.
2. Related Art
The aim of crop plant genetic engineering is to insert a gene (or genes) which alter a plant's characteristics without altering otherwise desirable elements of the genotype of the original plant. Thus, the genetic make-up of crop plants can be extended to include genes outside of the original genetic pool which are not accessible by traditional crop breeding techniques. One important aspect of such modification is the choice of promoter. Conceptually, identification and characterisation of promoters allows the possibility to construct chimeric genes in which a promoter from one gene can be used to drive the expression of a protein encoded by a separate gene under conditions and at sites within the plant specified by the promoter.
Promoters often contain elements which are recognised by inducible factors which regulate the temporal and tissue-specific expression of genes. These elements are typically short sequences and are found in promoters of many (if not all) genes which respond to the same signal. Thus, in plants, analysis of promoters of genes which are upregulated by the phytohormone abscisic acid have identified a common element CCACGTT within these promoters (Marcotte et al., Plant Cell 1 969-976 (1989) and Pla et al., Plant Mol. Biol. 21 259-266 (1993)). Similarly, promoters responsive to ethylene contain the PR box AGCCGCC (Broglie et al., Plant Cell 1 599-607 (1989) and Deickman et al., Plant Physiol. 100 2013-2017 (1992)). Furthermore, possession of a selection of such response elements may confer upon a promoter either the ability to drive gene expression in response to any one of several signals or to display synergistic enhancement of expression in response to several signals. Table 1 lists a number of consensus response element sequences identified in plants.
TABLE 1Promotor Response ElementsNameSequenceSensitivityABRECCACGTTABADRE1TACCGACATDroughtE-8ATAAGGGGTTGGT(SEQ ID NO:5)G BoxGTGTCACH BoxGGTAGGJA BoxCCCTATAGGGJA?(SEQ ID NO:6)MybTGGTTAMycCANNTGPR BoxAGCCGCCEthyleneTCATTATCTCCTT(SEQ ID NO;7)
Thus, from the DNA sequence of promoters, it is possible to predict the circumstances under which a promoter will be expressed by looking for already identified response elements within its sequence. It is with such inducible promoters that the present application is concerned.
Previously the expression of foreign proteins in transgenic plants has been driven by a ‘constitutive’ promoter such as Cauliflower mosaic virus 35S (CaMV 35S). Use of such a promoter in a commercial application is limited where the promoter is being used to drive toxic protein synthesis, or proteins which impose a substantial metabolic burden on the plant. These problems can be overcome if expression of the target protein is under the control of an inducible promoter, either just prior to or just after harvesting.
Several plant derived promoters have been previously proposed as being suitable for chemically regulated transgene expression (Gatz, C (1997) Annu. Rev. Plant Physiol. Plant. Mol. Biol. 48, 89-108). Such promoters are inducible by safeners or elicitors or chemicals mediating wound responses or chemicals inducing systemic acquired resistance (SAR). Promoters induced by safeners or by chemicals that induce SAR have been most studied. For example WO 93/01294 and Jepson et al., (1994) Plant Mol. Biol. 26, 1855-1866, describe the isolation of a glutathione S-transferase gene (GST-27) inducible by the safener N,N-diallyl-2,2-dichloroacetimide. The applications of this promoter are however limited since the promoter is constitutively expressed in roots.
The phenomenon of systemic acquired resistance (SAR) following infection of a plant with pathogenic micro-organisms has long been established. When a plant is invaded by a potential pathogen, which it is able to recognise, a resistance response is activated. This response, known as an incompatible interaction, typically involves hypersensitive cell death at the site of pathogen ingress, phytoalexin synthesis, the production of active oxygen species, cell wall strengthening, local induction of defence genes and salicylic acid (SA) accumulation. Following on from this local response, SAR is established throughout the plant. SAR endows uninfected tissue with the ability to respond more rapidly to further infection and this resistance is effective against a wide range of potential pathogens. The synthesis of a number of relatively unrelated proteins known as pathogenesis-related proteins (PR proteins) accompanies the onset and establishment of SAR.
It has long been known that treatment of plants with either SA or acetyl-SA (aspirin) can induce resistance to pathogens (White, R. F. (1979). Virology 99, 410-412). Recent research has demonstrated that SA plays a key role in both the local and systemic induction of PR proteins and the establishment of SAR (Malamy et al., (1990) Science 250, 1002-1004; Metraux et al., (1990) Science 250, 1004-1006; Yalpani et al., (1991): Plant Cell 3, 809-818). Furthermore, SAR and PR protein accumulation are compromised in plants that constitutively express a bacterial salicylate hydroxylase (which converts SA to catechol) further supporting a role for endogenous SA in these processes (Gaffney et al., (1993) Science 261, 754-756). Spraying, injection or root-feeding plants with SA strongly induces expression of PR gene promoters by 50-1000-fold over basal levels (e.g. Mur et al., (1996) Plant J. 9, 559-571). However, since the concentration of SA used in these studies (typically 1-2 mM) is phytotoxic, a great deal of effort has been put into identifying less harmful compounds capable of mimicking SA.
One compound in particular, BTH (benzo(1,2,3)thiadiazole-7-carbothoic acid S-methyl ester), is already marketed as a ‘crop enhancer’ and is available for large scale use in the field (Gorlach et al., (1996) Plant Cell 8, 629-643). An aqueous solution of 1.2 μM BTH is sufficient to induce PR gene expression (Friedrich et al., (1996) Plant J. 10, 61-70). Commercial preparations of BTH are sufficient to induce very strong PR gene expression in all plants tested including arabidopsis and wheat.
Gene induction following spraying with BTH is maximal 2 days after application and persists for at least 10 days. Although it induces enhanced resistance in treated tissue, its mode of action is unknown, nor is it known whether this compound can mimic all of SA's effects such as potentiation of gene induction or the pathogen-induced oxidative burst.
Thus, a potential source for inducible promoters is the pathogenesis-related (PR) ‘family’ of defence-related genes. PR genes are a diverse set of proteins some of which (e.g. PR-2 and PR-3 classes) have known functions as chitinases or beta-1,3-glucanase. Others (e.g. the PR-1 and PR-5 classes) are induced during plant-pathogen responses but have no clearly identifiable function. Typically, PR proteins of each class contain members with acidic or with basic pHs. Although there are exceptions to the rule, basic PR proteins tend to be localised to an intracellular site (e.g the vacuole) whilst acidic PR proteins are secreted.
Plants also have to respond to a variety of other environmental stresses including water stress, mechanical and herbivore wounding, UV light and oxidative stress, and both high and low temperatures. PR genes are upregulated in a number of these conditions. Thus, expression of tobacco osmotin (a basic, vacuolar PR-5 gene) is induced not only by pathogen challenge but also by salt stress (Grillo et al., Physiologia Plantarum 93 498-504 (1995)). PR-1a expression is induced following treatment with hydrogen peroxide (which induces oxidative stress) and in plants subjected to UV stress (Yalpani et al., Planta 193 372-376 (1994)). The responses to wounding and to pathogen challenge share a number of similar features including expression of defence genes and the establishment of a systemic response mediated by mobile signals. As a rule, basic PR proteins are also responsive to wounding stimuli.
A number of elements present in PR gene promoters have been identified. The PR-2d gene (encoding a β-1,3-glucanase) from tobacco is expressed in tissue undergoing hypersensitive response (HR) following tobacco mosaic virus (TMV) challenge and is induced by exogenous SA (Shah et al., Plant J. 10:1089-1101 (1996)). Region −364 to −288 in the PR-2d promoter confers SA sensitivity and a 25 bp element in this region is recognised by nuclear factors from tobacco. An SA responsive element has also been isolated from the CaMV 35S promoter at position −90 to −46. The element corresponds to an as-1 site (Qin et al., Plant Cell 6:863-874 (1994)). The sequence TCATCTTCTT (SEQ ID NO:8) is repeated several times in the barley β-1,3-glucanase promoter and is present in over 30 stress-induced genes (Goldsbrough et al., Plant J. 3(4):563-571 (1993b)). This region binds 40 kDa tobacco nuclear proteins, the binding of which is increased in SA-treated plants. Buttner et al., Proc. Natl. Acad. Sci. USA 94:5961-5966 (1997) have shown that Arabidopsis ethylene responsive element binding proteins bind to the PR box and that PR- and G-boxes exhibit synergistic effects.
PR-1 genes have been studied in some detail and the promoter of one, tobacco PR-1a, has been proposed as a suitable inducible promoter (EP 0 332 104 A2). Tobacco PR-1a is expressed both locally in infected tissue and later during establishment of SAR. Thus, infection of Samsun NN tobacco plants leads to accumulation of endogenous PR-1 proteins in both inoculated leaves (approx. 4 days after infection) and later (approx. 8 days) in upper uninfected leaves on the same plant. Local (approx. 12 hours post-inoculation) and systemic (3-7 days) induction of PR-1a-GUS expression in Pseudomonas syringae pathovar syringae-infected tobacco has been reported. (Bi et al., (1995) Plant J. 8:235-245; Mur et al., (1996) Plant J. 9:559-571). Direct application of SA induces high levels of PR-1a promoter-GUS expression in transgenic tobacco [Bi et al., supra]. The SAR inducers BTH and INA also induce high levels of both endogenous PR-1a and PR-1a-GUS expression.
Wounding also induces a slight increase in PR-1a-GUS expression (Darby, R., unpublished observations, Ohshima et al., (1990) Plant Cell 2, 95-106). As with other PR-1 proteins, PR-1a exhibits developmental expression. Thus, PR-1a-GUS is expressed in leaves, petioles, stem cortex, pollen and sepals of flowering tobacco (Uknes et al., Plant Cell, 5(2):159-169 (1993)). PR-1a-GUS is also expressed in roots (Kenton, P; unpublished observations).
The PR-1a promoter has been studied extensively. Van de Rhee and Bol (Plant Mol. Biol., 21(3):451-461 (1993b)) identified four regulatory elements in the PR-1a promoter all of which were required for maximal activity and no single element of which was capable of conferring promoter activity. The PR-1a promoter contains a number of elements that bind GT-1-like and Myb1 transcription factors (Buchel et al., Plant Mol. Biol., 30(3):493-504 (1996)). In addition, SA and active analogues induce expression not only of PR genes but also myb1.
The high level of sensitivity to SA shown by the PR-1a gene and the very high levels of PR-1α-GUS expression following SA treatment or infection could lead to inappropriate expression of any PR-1a promoter-gene fusion as a result of perturbation of endogenous SA levels (brought about, e.g., by a change in redok status). This may limit its usefulness in driving genes the products of which are either toxic at high levels or impose a substantial metabolic burden on the plant. Finally, PR-1 genes in general and PR-1a in particular show a high level of constitutive/developmental expression, especially during flowering. Again, this could lead to a high degree of unscheduled expression of PR-1a promoter-driven transgenes.
PR-5 proteins are another class of PR proteins, and can be divided into two groups, the acidic extracellular thaumatin-like proteins and the basic intracellular osmotins. Classically osmotins have been associated with abiotic stresses. However, this osmotically-induced expression is typically additional to a high degree of constitutive (Stintzi et al., Biochimie, 75:687-706 (1993); Leone et al., Plant Physiol., 106:703-712 (1994); Van Kan et al., Plant Mol. Biol., 27:1205-1213 (1995)) and developmental expression (Linthorst, Crit. Rev. Plant Sci., 10:123-150 (1991); Stintzi et al., Physiol. Mol. Plant Pathol. 38 137-146 (1991); Raghothama et al., Plant Mol. Biol. 34:393-402 (1997)). Osmotin expression is also elevated in response to stresses such as desiccation, wounding, low temperature (Raghothama et al., Plant Mol. Biol. 23:1117-1128 (1993); Grillo et al., (1995) supra; Zhu et al., Plant Mol. Biol., 28:17-26 (1995b)), and chemical factors such as ethylene (in tobacco Raghothama et al., 1993, supra; Chang et al., Physiologia-Plantarum, 100:341-352 (1997)), and cytokinins (Thomas & Bohnert, Plant Physiol., 103:1299-1304 (1993)). Pathogen challenge also induces osmotin expression (Zhu et al., Plant Physiol., 108:929-937 (1995a); (1995b), supra; Chang et al., (1997), supra) which may be systemic for some osmotins (Zhu et al., (1995b), supra) or local for others (Zhu et al., (1995a), supra). Thus the osmotin genes do not appear to be ideal sources of inducible promoters.
Unlike the vacuolar-localised osmotins, acidic PR-5s (aPR-5) are secreted and lack the C-terminal extension which may be a vacuolar targeting signal (Linthorst, (1991), supra; Stintzi et al., (1993), supra). aPR5 proteins have been shown to be accumulated on pathogen attack, for example in barley (Bryngelsson & Green, Plant Mol. Plant Path., 35:45-52 (1989); Boyd et al., Plant Mol. Plant Path., 45:47-58 (1994);Reiss & Bryngelsson, Physiol. Mol. Plant Path., 43:331-341 (1996);Schweizer et al., Plant Physiol., 114:73-88 (1997);Vale et al., Physiol. Mol. Plant Path., 44:207-215 (1994)) and wheat (Rebmann et al., Plant Mol. Biol., 17:283-285 (1991)). Using western blots, Stintzi et al., (1991) were unable to detect aPR-5 in healthy tobacco leaves whereas osmotin was constitutively expressed. Following challenge with TMV, aPR-5 appears after 4-6 hours, whereas osmotin begins to accumulate over basal levels 2-4 hours post-inoculation (Stintzi et al., 1991). aPR-5 has been localised to extracellular pocket-like structures between mesophyll cells close to the infection site in TMV-infected tobacco (Dore et al., Arch. Virol., 120:97-107 (1991)).
A number of other treatments have been shown to induce expression of extracellular aPR-5 proteins. Sunflower extracellular aPR-5 proteins are induced in leaf discs by 5 mM aspirin, 10 mM ethephon, 10 mM NAA, 10 mM 2,4 D, UV light, 5 mM MnCl2, 5 mM HgCl3, 5 mM citric acid and 5 mM oxalic acid (Jung et al., Journal of Plant Physiol., 145:153-160 (1995)). 1 ppm INA induces expression of a barley homologue of rice thaumatin-like protein and JA that of a barley aPR5 (Schweizer et al., (1997), supra). aPR-5s are also expressed in cold-acclimated winter rye where they may play a role in preventing ice damage (Hon et al., Plant Physiol., 109:879-889 (1995)).
However, information concerning developmental expression of aPR-5s is limited. In maize constitutive expression is mainly confined to non-embryonic tissues of the developing seed peaking two to four weeks after pollination but still detectable in desiccated seed. Only slight expression was detectable in maize leaves (Malehorn et al., Plant Physiol., 106:1471-1481 (1994)). A 29 kDa thaumatin-like protein has been detected in ripe cherry fruits (Fils-Lycaon et al., Plant Physiol., 111:269-273 (1996)).
Little is known about aPR-5 promoters since only a tobacco aPR5 promoter from the E2 gene has been isolated, fused to the reporter gene GUS and analysed (Albrecht et al., (1992) Plant Mol Biol. 18, 155-158). This study showed that TMV induced both local and systemic GUS activity; the local response being greater than the systemic response. The element(s) responsible for this TMV induction of this aPR-5 were found to lie in the −1364 to −718 promoter region. By nucleic acid hybridisation no significant homology was found between this tobacco PR-5 promoter and the PR-1a promoter.